Membrane lytic poly(amido amine) polymers for the delivery of oligonucleotides

ABSTRACT

The present invention provides membrane lytic poly(amido amine) polymers, polyconjugates, compositions and methods for the delivery of oligonucleotides for therapeutic purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is being submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “MRLMIS00043USPCT-SEQTXT-17MAY2013.txt”, creation date of May17, 2013 and a size of 4.52 KB. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Oligonucleotides conjugated to polymers are known. Further, the deliveryof oligonucleotides conjugated to polymers (polyconjugates) fortherapeutic purposes is also known. See WO2000/34343; WO2008/022309; andRozema et al. PNAS (2008) 104, 32: 12982-12987.

Poly(amido amine) polymers are known. Z. Khayat et al. InternationalJournal of Pharmaceutics (2006) 317:175-186; P. Ferruti et al. Macromol.Rapid Commun. (2002) 23:332-355; and Ka-Wai Wan et al. Biomacromolecules(2004) 5:1102-1109.

Polyconjugates have numerous toxicities associated with it as anoligonucleotide delivery vehicle. It is thus an object of the inventionto provide polyconjugates that are biodegradable. Further, it is also anobject of the invention to provide polyconjugates designed withcomponents to increase liver uptake. Further, it is also an object ofthe instant invention to provide polyconjugates designed with componentsthat facilitate oligonucleotide escape from the endosome. Herein, wedisclose and describe novel membrane lytic poly(amido amine) polymersand polyconjugates useful for the delivery of oligonucleotides fortherapeutic purposes.

SUMMARY OF THE INVENTION

The present invention provides membrane lytic poly(amido amine)polymers, polyconjugates, compositions and methods for the delivery ofoligonucleotides for therapeutic purposes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. RBC Hemolysis Data of Polyconjugates

FIG. 2. Liver Apo B mRNA-Mice

FIG. 3. Liver Apo B mRNA-Mice

FIG. 4. Liver Apo mRNA and ALT-Rats

FIG. 5. NMR spectra for the synthesized poly (amido amine) “PPA”polymers recorded on varian spectrometer operating at 500 MHz.

FIG. 6. The molecular weight and polydispersity (Mw/Mn) of thesynthesized poly (amido amine) “PPA” polymers determined by GPC.

FIG. 7. GPC calibration plot and GPC calibration table for thesynthesized poly (amido amine) “PPA” polymers.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the instant invention is a polymer comprising FormulaZ:

wherein:

n is 2 to 250;

R¹ is independently selected from a primary, secondary, tertiary andquaternary amine; and

R² is independently selected from a primary, secondary, tertiary andquaternary amine, a heterocyclic amine, and a lipophilic group; or astereoisomer thereof.

Another embodiment of the instant invention is a polymer comprisingFormula Z′:

wherein:

n is 2 to 250;

R is a capped end group selected from a primary and secondary amine;

R′ is hydrogen or methylene;

R¹ is independently selected from a primary, secondary, tertiary andquaternary amine; and

R² is independently selected from a primary, secondary, tertiary andquaternary amine, a heterocyclic amine, and a lipophilic group;

or a stereoisomer thereof.

In another embodiment of the instant invention is a polymer comprisingFormula Z′, wherein:

n is 2 to 250;

R is C₄H₉NH;

R′ is hydrogen;

R¹ is aminoethoxy; and

R² is independently selected from dodecyl, 2-(1H-imidazol-4-yl)ethyl and2-(2-aminoethoxy)ethyl;

or a stereoisomer thereof.

In another embodiment of the instant invention is a polymer conjugatecomposition comprising a polymer of Formula Z or Z′, a linker and anoligonucleotide.

In another embodiment of the instant invention is the polymer conjugatecomposition above further comprising a masking agent.

In another embodiment of the instant invention is the polymer conjugatecomposition above further comprising a targeting ligand.

In another embodiment of the instant invention is the polymer conjugatecomposition above further comprising a masking agent and a targetingligand.

In another embodiment of the instant invention is a polymer conjugatecomposition made by the 1) synthesis of an activated polymer comprisingFormula Z or Z′; 2) synthesis of an activated oligonucleotide; and 3)conjugation of the activated polymer with the activated oligonucleotide;optionally including the addition of a masking agent and/or a targetingligand.

In another embodiment of the instant invention is a method of treating adisease in a patient by administering a polymer conjugate composition ofthe instant invention.

Definitions

“Amine (primary, secondary, tertiary or quaternary)” means organiccompounds and functional groups that contain a basic nitrogen atom witha lone pair. Amines are derivatives of ammonia, wherein one or morehydrogen atoms have been replaced by a substituent such as an alkyl oraryl group.

“Capped end group(s)” means the terminal end group(s) of polymers thatinhibit the ability of the polymer to continue polymeriazation.

“Heterocyclic amine” means an organic compound containing at least oneatom of carbon and at least one atom on nitrogen within a ringstructure. These structures may comprise either simple aromatic rings ornon-aromatic rings.

“Disease” means a disorder or incorrectly functioning organ, part,structure, or system of the body resulting from the effect of genetic ordevelopmental errors, infection, poisons, nutritional deficiency orimbalance, toxicity, or unfavorable environmental factors; illness;sickness; ailment. An example of a disease is cancer.

“Lipophilic group” means groups having affinity for lipids. Lipophilicsubstances interact within themselves and with other substances throughthe dispersion force and they have little to no capacity to formhydrogen bonds.

“Linker” means a chemical moiety that physically conjugates theoligonucleotide with a polymer comprising Formula Z or Z′.

“Masking agent” means a molecule which, when linked to a polymer,shields, inhibits or inactivates one or more properties (biophysical orbiochemical characteristics) of the polymer. Masking agents may beattached covalentely to targeting ligands or polyethylene glycol. SeeWO2008/022309 for a more detailed description of masking agents.

“Oligonucleotide” means deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) and combinations of DNA, RNA and other natural and syntheticnucleotides, including protein nucleic acid (PNA). DNA maybe in form ofcDNA, in vitro polymerized DNA, plasmid DNA, parts of a plasmid DNA,genetic material derived from a virus, linear DNA, vectors (P1, PAC,BAC, YAC, and artificial chromosomes), expression vectors, expressioncassettes, chimeric sequences, recombinant DNA, chromosomal DNA,anti-sense DNA, or derivatives of these groups. RNA may be in the formof messengerRNA (mRNA), in vitro polymerized RNA, recombinant RNA,transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA),chimeric sequences, anti-sense RNA, interfering RNA, small interferingRNA (siRNA), microRNA (miRNA), ribozymes, external guide sequences,small non-messenger RNAs (snmRNA), untranslatedRNA (utRNA), snoRNAs(24-mers, modified snmRNA that act by an anti-sense mechanism), tinynon-coding RNAs (tncRNAs), small hairpin RNA (shRNA), or derivatives ofthese groups. In addition, DNA and RNA may be single, double, triple, orquadruple stranded. Double, triple, and quadruple strandedpolynucleotide may contain both RNA and DNA or other combinations ofnatural and/or synthetic nucleic acids. Oligonucleotides can bechemically modified. The use of chemically modified oligonucleotides canimprove various properties of the oligonucleotides including, but notlimited to: resistance to nuclease degradation in vivo, cellular uptake,activity, and sequence-specific hybridization. Non-limiting examples ofsuch chemical modifications include: phosphorothioate internucleotidelinkages, LNA, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, 2′-deoxy ribonucleotides, “universal base” nucleotides,5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation.These chemical modifications, when used in various oligonucleotideconstructs, are shown to preserve oligonucleotide activity in cellswhile at the same time, dramatically increasing the serum stability ofthese compounds. Chemically modified siRNA can also minimize thepossibility of activating interferon activity in humans. SeeWO2008/022309 for a more detailed description of oligonucleotides.

“Polyethylene glycols” (PEGs) are non-toxic and non-immunogenicpolyether compounds. They can be added to media and attached to surfacesand conjugated to molecules without interfering with cellular functionsor target immunogenicities. When attached to biomolecules PEGs decreaseaggregation and increase aqueous solubility.

“Polymer” means a molecule built up by repetitive bonding together ofsmaller units called monomers. A polymer can be linear, branched,network, star, comb, or ladder type. A polymer can be a homopolymer inwhich a single monomer is used or a polymer can be copolymer in whichtwo or more different monomers are used. Copolymers may by alternating,random (statistical), gradient, block and graft (comb). The monomers inrandom copolymers have no definite order or arrangement along any givenchain. The general compositions of such polymers are reflective of theratio of input monomers. However, the exact ratio of one monomer toanother may differ between chains. The distribution of monomers may alsodiffer along the length of a single polymer. Also, the chemicalproperties of a monomer may affect its rate of incorporation into arandom copolymer and its distribution within the polymer. Thus, whilethe ratio of monomers in a random polymer is dependent on the inputratio of monomer, the input ratio may not match exactly the ratio ofincorporated monomers. See WO2008/022309 for a more detailed descriptionof polymers.

“Patient” means a mammal, typically a human, in need of treatment for adisease.

“Purification step” means a process by which a desired compound isseparated from a mixture of other compounds.

“Tangential Flow Filtration (TFF)” means a rapid and efficient methodfor filtration and separation of solutions containing large molecules,biomolecules, or particles such as viruses, bacteria or cellularmaterial. It is a process whereby product flow (feed) is directedtangentially along the surface of a membrane with most of the solutioncirculated back to the feed tank. The rapid flow of feed solution acrossthe membrane acts to ‘sweep’ the surface, reducing concentrationpolarization (product concentration at the membrane surface). It alsoprevents build-up of foulants that can plug the pores at the membranesurface. The rapid cross flow creates a pressure drop, which forces someof the feed solution and dissolved molecules that are smaller than thepores in the membrane, through the membrane filter. The solution thatpasses through the membrane is referred to as filtrate or permeate.Molecules or particles larger than the membrane pores are retained inthe feed solution and effectively concentrated.

“Targeting ligand”, also referred to as “targeting agent”, means anagent that can deliver a polymer or polyconjugate to target cells ortissues, or specific cells types. Targeting ligands enhance theassociation of molecules with a target cell. Thus, targeting ligands canenhance the pharmacokinetic or biodistribution properties of apolyconjugate to which they are attached to improve cellulardistribution and cellular uptake of the conjugate. See WO2008/022309 fora more detailed description of targeting ligands.

In an embodiment of Formula Z or Z′, n is 2 to 250.

In another embodiment of Formula Z or Z′, n is 2 to 225.

In another embodiment of Formula Z or Z′, n is 2 to 200.

In another embodiment of Formula Z or Z′, n is 2 to 175.

In another embodiment of Formula Z or Z′, n is 2 to 150.

In another embodiment of Formula Z or Z′, n is 2 to 125.

In another embodiment of Formula Z or Z′, n is 2 to 100.

In another embodiment of Formula Z or Z′, n is 2 to 75.

In another embodiment of Formula Z or Z′, n is 2 to 50.

In another embodiment of Formula Z or Z′, n is 2 to 25.

In an embodiment of Formula Z or Z′, n is 5 to 250.

In another embodiment of Formula Z or Z′, n is 5 to 225.

In another embodiment of Formula Z or Z′, n is 5 to 200.

In another embodiment of Formula Z or Z′, n is 5 to 175.

In another embodiment of Formula Z or Z′, n is 5 to 150.

In another embodiment of Formula Z or Z′, n is 5 to 125.

In another embodiment of Formula Z or Z′, n is 5 to 100.

In another embodiment of Formula Z or Z′, n is 5 to 75.

In another embodiment of Formula Z or Z′, n is 5 to 50.

In another embodiment of Formula Z or Z′, n is 5 to 25.

In another embodiment of Formula Z or Z′, n is 10 to 60.

In another embodiment of Formula Z or Z′, n is 15 to 55.

In another embodiment of Formula Z or Z′, n is 20 to 50.

In another embodiment of Formula Z or Z′, n is 25 to 45.

In another embodiment of Formula Z or Z′, n is 30 to 40.

In an embodiment R is C₄H₁₀N.

In an embodiment R′ is hydrogen.

In an embodiment, R¹ is independently selected from a primary,secondary, tertiary and quaternary amine.

In another embodiment, R¹ is independently selected from aminoethoxy,2-(2-aminoethoxy)ethyl, 2-[2-2-aminoethoxy)ethoxy]ethyl,2-[2-(2-aminoethoxy)ethoxy]ethyl, 3-amino-2-hydroxypropyl, 2-aminoethyl,4-aminobutyl, 6-aminohexyl, 8-aminooctyl and 10-aminodecyl.

In another embodiment, R¹ is aminoethoxy.

In an embodiment, R² is independently selected from a primary,secondary, tertiary and quaternary amine, a heterocyclic amine, and alipophilic group.

In another embodiment, R² is independently selected from2-(2-aminoethoxy)ethyl, 2-[2-(2-aminoethoxy)ethoxy]ethyl,2-[2-(2-aminoethoxy)ethoxy]ethyl, 3-amino-2-hydroxypropyl, 2-aminoethyl,4-aminobutyl, 6-aminohexyl, 8-aminooctyl, 10-aminodecyl,2-(1H-imidazol-4-yl)ethyl, 2-(4-methyl-1H-imidazol-5-yl)ethyl,2-(1-Ethyl-1H-imidazol-4-yl)-ethyl, 2-(5-Methyl-3H-imidazol-4-yl)-ethyl,2-(2-isopropyl-1-methyl-1H-imidazol-4-yl)ethyl,2-(1-butyl-1H-imidazol-4-yl)ethyl, 2-(1-hexyl-1H-imidazol-4-yl)ethyl,2-(1-octyl-1H-imidazol-4-yl)ethyl, 2-(1-dodecyl-1H-imidazol-4-yl)ethyl,2-pyridin-4-yl ethyl, 2-(2,6-dimethylpyridin-4-yl)ethyl, 2-pyridin-2-ylethyl, 2-pyridin-3-yl ethyl, 2-piperazin-1-yl ethyl,[4-(2-ethyl)piperidin-1-yl]methanol and 2-morpholin-4-ylethyl.

In another embodiment, R² is independently selected from2-(1H-imidazol-4-yl)ethyl, 2-(4-methyl-1H-imidazol-5-yl)ethyl,2-(1-Ethyl-1H-imidazol-4-yl)-ethyl, 2-(5-Methyl-3H-imidazol-4-yl)-ethyl,2-(2-isopropyl-1-methyl-1H-imidazol-4-yl)ethyl,2-(1-butyl-1H-imidazol-4-yl)ethyl, 2-(1-hexyl-1H-imidazol-4-yl)ethyl,2-(1-octyl-1H-imidazol-4-yl)ethyl, 2-(1-dodecyl-1H-imidazol-4-yl)ethyl,2-pyridin-4-yl ethyl, 2-(2,6-dimethylpyridin-4-yl)ethyl, 2-pyridin-2-ylethyl, 2-pyridin-3-yl ethyl, 2-piperazin-1-yl ethyl,[4-(2-ethyl)piperidin-1-yl]methanol and 2-morpholin-4-ylethyl.

In another embodiment, R² is independently selected from a lipophilicgroup which is selected from an alkyl group, an alkenyl group and analkynyl group, all of which may be branched or cyclic or acyclic oraromatic.

In an embodiment, a linker is the chemical moiety which is made by theconjugation of a derivative of SMPT (4-succinimidyloxycarbonyl-

-methyl-

-[2-pyridyldithio]toluene) and/or a derivative of SATA(N-Succinimidyl-S-acetylthioacetate).

In an embodiment, a masking agent is selected from a maleic anhydridederivative.

In an embodiment, a masking agent is selected from a disubstitutedmaleic anhydride derivative.

In an embodiment, a targeting ligand is selected from compounds withaffinity to cell surface molecules, cell receptor ligands, andantibodies, antibody fragments, and antibody mimics with affinity tocell surface molecules.

In another embodiment, a targeting ligand is selected fromcarbohydrates, glycans, saccharides (including, but not limited to:galactose, galactose derivatives, mannose, and mannose derivatives),vitamins, folate, biotin, aptamers, and peptides (including, but notlimited to: RGD-containing peptides, insulin, EGF, and transferrin).

In another embodiment, a targeting ligand is selected fromN-acetylgalactosamine (NAG), mannose and glucose.

In another embodiment, a targeting ligand is N-acetylgalactosamine(NAG).

In an embodiment, an oligonucleotide is selected from siRNA, miRNA andantisense. In another embodiment, an oligonucleotide is an siRNA.

In an embodiment, the polymers of Formula Z or Z′ are copolymers.

In an embodiment, the polymers of Formula Z or Z′ are copolymers whichare random.

In an embodiment, the polymers of Formula Z or Z′ are copolymers whichare gradient.

In an embodiment, the polymers of Formula Z or Z′ are copolymers whichare block.

Formulation

The polyconjugate, also known as “polymer conjugate” (composition of thepolymer comprising Formula Z or Z′ and an oligonucleotide) is formed bycovalently linking the oligonucleotide to the polymer. Conjugation ofthe oligonucleotide to the polymer can be performed in the presence ofan excess of polymer. Because the oligonucleotide and the polymer may beof opposite charge during conjugation, the presence of excess polymercan reduce or eliminate aggregation of the polyconjugate. Excess polymercan be removed from the polyconjugate prior to administration of thepolyconjugate to a patient. Alternatively, excess polymer can beco-administered with the polyconjugate to the patient.

Similarly, the polymer can be conjugated to a masking agent in thepresence of an excess of polymer or masking agent. Because theoligonucleotide and the polymer may be of opposite charge duringconjugation, the presence of excess polymer can reduce or eliminateaggregation of the polyconjugate. Excess polymer can be removed from thepolyconjugate prior to administration of the polyconjugate to a patient.Alternatively, excess polymer can be co-administered with thepolyconjugate to the patient. The polymer can be modified prior to orsubsequent to conjugation of the oligonucleotide to the polymer.

Similarly, the polymer can be conjugated to a targeting ligand in thepresence of an excess of polymer or targeting ligand. Because theoligonucleotide and the polymer may be of opposite charge duringconjugation, the presence of excess polymer can reduce or eliminateaggregation of the polyconjugate. Excess polymer can be removed from thepolyconjugate prior to administration of the polyconjugate to a patient.Alternatively, excess polymer can be co-administered with thepolyconjugate to the patient. The polymer can be modified prior to orsubsequent to conjugation of the oligonucleotide to the polymer.

Similarly, the polymer can be conjugated to a PEG in the presence of anexcess of polymer. Because the oligonucleotide and the polymer may be ofopposite charge during conjugation, the presence of excess polymer canreduce or eliminate aggregation of the polyconjugate. Excess polymer canbe removed from the polyconjugate prior to administration of thepolyconjugate to a patient. Alternatively, excess polymer can beco-administered with the polyconjugate to the patient. The polymer canbe modified prior to or subsequent to conjugation of the oligonucleotideto the polymer.

Parenteral routes of administration include intravascular (intravenous,interarterial), intramuscular, intraparenchymal, intradermal, subdermal,subcutaneous, intratumor, intraperitoneal, intrathecal, subdural,epidural, and intralymphatic injections that use a syringe and a needleor catheter. Intravascular herein means within a tubular structurecalled a vessel that is connected to a tissue or organ within the body.Within the cavity of the tubular structure, a bodily fluid flows to orfrom the body part. Examples of bodily fluid include blood,cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vesselsinclude arteries, arterioles, capillaries, venules, sinusoids, veins,lymphatics, bile ducts, and ducts of the salivary or other exocrineglands. The intravascular route includes delivery through the bloodvessels such as an artery or a vein. The blood circulatory systemprovides systemic spread of the pharmaceutical. An administration routeinvolving the mucosal membranes is meant to include nasal, bronchial,inhalation into the lungs, or via the eyes. Intraparenchymal includesdirect injection into a tissue such as liver, lung, heart, muscle(skeletal muscle or diaphragm), spleen, pancreas, brain (includingintraventricular), spinal cord, ganglion, lymph nodes, adipose tissues,thyroid tissue, adrenal glands, kidneys, prostate, and tumors.Transdermal routes of administration have been affected by patches andiontophoresis. Other epithelial routes include oral, nasal, respiratory,rectum, and vaginal routes of administration.

The polyconjugates can be injected in a pharmaceutically acceptablecarrier solution. Pharmaceutically acceptable refers to those propertiesand/or substances which are acceptable to the patient from apharmacological/toxicological point of view. The phrase pharmaceuticallyacceptable refers to molecular entities, compositions, and propertiesthat are physiologically tolerable and do not typically produce anallergic or other untoward or toxic reaction when administered to apatient. Preferably, as used herein, the term pharmaceuticallyacceptable means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

Utility

The polyconjugates (compositions of a polymer comprising Formula Z or Z′and an oligonucleotide) of the instant invention may be used forresearch purposes or to produce a change in a cell that can betherapeutic. The use of polyconjugates for therapeutic purposes isknown. See WO2000/34343; WO2008/022309; and Rozema et al. PNAS (2008)104, 32: 12982-12987.

EXAMPLES

Examples and schemes provided are intended to assist in a furtherunderstanding of the invention. Particular materials employed, speciesand conditions are intended to be further illustrative of the inventionand not limitative of the reasonable scope thereof.

Monomer Synthesis

Novel tert-butyl(2-{[1,3-bis(prop-2-enoylamino)propan-2-yl]oxy}ethyl)carbamate (8) wassynthesized to increase the amine density of polymer. The monomer wassynthesized in seven steps.

Step 1

A 20-L 4-neck round-bottom flask was flushed with N₂. Then charged asolution of 1,3-bis(benzyloxy)propan-2-ol (2632 g, 9.7 mol, 1.2 eq) intetrahydrofuran (8400 mL, 3.2 v), N,N-dibenzyl-2-chloroethanaminiumchloride (2422 g, 8.2 mol, 1.0 eq), tetrabutylammonium bromide (1020 g,3.2 mol, 0.4 eq) and a solution of potassium hydroxide (2290 g, 40.9mol, 5.0 eq) in water (8400 mL, 3.2 v). The resulting solution wasstirred overnight at 65˜70° C. The reaction progress was monitored withLCMS. After the reaction was finished, the system was cooled to 20˜30°C. and then extracted with dichloromethane (2×4000 mL). The combinedorganic layer was washed with HCl (1M, 2×10 L). Adjusted the organiclayer to pH 8 with saturated aq. sodium bicarbonate. The separatedorganic layer was dried and concentrated under vacuum. The residue wasapplied onto a silica gel column and eluted with ethyl acetate/petroleumether (1:100). This resulted in 2888 g (71%) ofN,N-dibenzyl-2-(1,3-bis(benzyloxy)propan-2-yloxy)ethanamine as a yellowoil.

Step 2

Charged a solution of N,N-dibenzyl-2-(1,3-bis(benzyloxy)propan-2-yloxy)ethanamine (2000 g, 4.0 mol, 1.0 eq) in EtOH (10000 mL, 5.0 v),Pd/C (10%, 400 g, 20% wt,) and acetic acid (970 g, 16.2 mol, 4.0 eq) toa 20 L pressure reactor. The resulting mixture was stirred for 10 hoursat 80° C. under hydrogen (20 atm). The reaction progress was monitoredwith NMR. The reaction mixture was cooled to 20˜30° C. and filtered. HCl(36%, 500 ml, 6.0 mol, 1.5 eq) was added to the filtrate, stirred for 10minutes and concentrated under vacuum to give 572 g (83.4%) of2-(2-aminoethoxy)propane-1,3-diol hydrochloride as an oil. H-NMR of 3(300 MHz, D₂O, ppm): 3.79 (2H, q), 3.60 (2H, m), 3.50 (3H, m), 3.15 (2H,q).

Step 3

A 5-L 4-neck round-bottom flask was flushed with N₂. Then charged asolution of 2-(2-aminoethoxy)propane-1,3-diol (97.2 g, 0.7 mol, 1.0 eq)in methanol (1798 mL, 18.5 v), di-tert-butyl dicarbonate (314.4 g, 1.4mol, 2.0 eq) and a solution of potassium (88.9 g, 1.6 mol, 2.2 eq) inwater (1798 mL, 18.5 v). The resulting solution was stirred for 3.5 h at17˜21° C. in a water bath. The resulting mixture was washed withpetroleum ether (1400.0 mL+1200.0 ml) and concentrated under vacuum. Theresidual solution was extracted with dichloromethane (6×500 mL). Thecombined organic layer was dried over anhydrous sodium sulfate andconcentrated under vacuum to give 79.1 g (59.4%) of tert-butyl2-(1,3-dihydroxypropan-2-yloxy)ethylcarbamate as a light yellow oil.

H-NMR of 4 (300 MHz, CDCl₃, ppm): 3.74 (6H, m), 3.51 (1H, m), 3.35 (2H,m), 1.47 (9H, s).

Step 4

A 20-L 4-neck round-bottom flask was flushed with N₂, then charged asolution of tert-butyl 2-(1,3-dihydroxypropan-2-yloxy)ethylcarbamate(418 g, 1.8 mol, 1.0 eq) in dichloromethane (9000 mL, 21.5 v) andN,N-diisopropylethylamine (574 g, 4.4 mol, 2.5 eq). This is followed bydropwise addition of methanesulfonyl chloride (509 g, 4.4 mol, 2.5 eq)with stirring at −6-0° C. over 1 hour. The resulting mixture was stirredfor 4 h at −6˜4° C., then washed with water (3×4000 mL). The organiclayer was separated, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was applied onto a silica gelcolumn and eluted with petroleum ether/EtOAc (1:1) to give 740 g(101.0%) of 2-(2-(tert-butoxycarbonylamino)ethoxy) propane-1,3-diyldimethanesulfonate as a yellow oil.

H-NMR of 5 (300 MHz, CDCl₃, ppm): 5.05 (1H, s, w), 4.24 (4H, m), 3.81(1H, m), 3.63 (2H, m), 3.24 (2H, m), 3.02 (6H, s), 1.37 (9H, s).

Step 5

A 20 L 4-neck round-bottom flask was flushed with N₂. Then charged asolution of 2-(2-(tert-butoxycarbonylamino) ethoxy)propane-1,3-diyl-dimethanesulfonate (929 g, 2.37 mol, 1.0 eq) inN,N-dimethylformamide (9300 mL) and potassium phthalimide (2194 g, 11.85mol, 5.0 eq). The resulting mixture was stirred for 18 hours at 70˜75°C. in an oil bath. The reaction progress was monitored with LCMS. Thereaction mixture was cooled to 20˜30° C., then added icy water (28 L)with stirring. The mixture was stirred for 30 minutes, and thenfiltered. The filter cake was dried under vacuum at 60° C. to give 919 g(78.5%) tert-butyl2-(1,3-bis(1,3-dioxoisoindolin-2-yl)propane-2-yloxy)ethylcarbamate as anoff-white solid.

LCMS of 6 (ES, m/z): 393.8 [M-Boc]⁺

Step 6

A 20 L 4-neck round-bottom flask was flushed with N₂. Then charged asolution of tert-butyl2-(1,3-bis(1,3-dioxoisoindolin-2-yl)propan-2-yloxy) ethylcarbamate (919g, 1.9 mol, 1.0 eq) in ethanol (12000 mL, 13.0 v), followed by dropwiseaddition of hydrazine hydrate (602 g, 12.0 mol, 6.3 eq) with stirring at40-45° C. over 30 min. The resulting mixture was stirred for 7 hours at40-45° C. in an oil bath. The reaction progress was monitored with LCMS.The reaction mixture was cooled to 20˜30° C. and filtered. The filtratewas concentrated under vacuum to give 424.0 g (97.5%) of tert-butyl2-(1,3-diaminopropan-2-yloxy) ethylcarbamate as a brown oil.

LCMS of 7 (ES, m/z): 234 [M+H]⁺

H-NMR of 7 (300 MHz, CDCl₃, ppm): 5.39 (1H, s, w), 3.60 (2H, q), 3.26(3H, m), 2.79 (4H, m), 1.43 (9H, s), 1.39 (4H, s)

Step 7

A 5 L 4-neck round-bottom flask was flushed with N₂ and kept from light.Then charged a solution of tert-butyl2-(1,3-tert-butyl2-(1,3-diaminopropan-2-yloxy)ethylcarbamate (300.0 g,1.3 mol, 1.0 eq) in dichloromethane (3000 mL, 10.0 v) andN-ethyl-N-isopropylpropan-2-amine (332.3 g, 2.6 mol, 2.0 eq), followedby dropwise addition of acryloyl chloride (232.7 g, 2.6 mol, 2.0 eq)with stirring at <3° C. over 150 min. The resulting mixture was stirredfor 90 min at 20-24° C. in a water bath. The reaction was monitored withLCMS. The resulting mixture was washed with water (1 L). The organiclayer was dried over anhydrous sodium sulfate and concentrated undervacuum. Heptane (6000 ml) was added dropwise with stirring to theresidue over 20 minutes, then cooled to 0° C. and stirred for 30minutes. The solid was collected and washed with hepatane (2×200 mL) togive 218.0 g (48.0%) of tert-butyl2-(1,3-diacrylamidopropan-2-yloxy)ethylcarbamate as an off-white solid.

LCMS of 8 (ES, m/z): 341.9[M⁺].

H-NMR of 8 (300 MHz, CDCl₃, ppm): 6.93 (2H, s, w), 6.25 (4H, m), 5.68(2H, dd), 3.65 (5H, m), 3.30 (4H, m), 1.46 (9H, s).

General Polymer Synthesis (Scheme 1)

The monomers were weighed and brought up in 30% ethanol solution inwater. The reaction mixture was stirred at 55° C. in dark for 5 daysunder nitrogen atmosphere. After 5 days, polymerization was quenched byadding 20 mol % excess of amine to consume any unreacted acrylate. Afterthat, polymer was precipitated with diethylether and dried. Polymerswith Boc-protected oligoamines were deprotected by TFA. The crudepolymer was precipitated again in diethylether. The polymer was furtherpurified by dialysis.

Polymer Synthesis (Scheme 2)

Poly (amido amine) “PAA” polymers were synthesized by polyaddition ofprimary amines to tert-butyl(2-{[1,3-bis(prop-2-enoylamino)propan-2-yl]oxy}ethyl)carbamate.

In a typical experiment, 8 (1 equiv: 0.585 mmol, 200 mg), tert-butyl[2-(2-aminoethoxy)ethyl]carbamate (0.4 equiv: 0.234 mmol, 47.8 mg),histamine (0.3 equiv: 0.175 mmol, 19.5 mg) and dodecylamine (0.3 equiv:0.175 mmol, 32.57 mg) were added to a reaction flask. The solventmixture (30% ethanol in water 1.2 ml; 0.5 M) was added to the reactionflask and the reaction mixture was degassed. Polymerization was carriedout in dark at 55° C. under nitrogen atmosphere. The reaction mixturebecame homogeneous in less than 15 minutes at 55° C. The reaction wasallowed to proceed for 5 days. Subsequently, 10-20 mol % of butylaminein EtOH was added to the reaction mixture to consume any unreactedacrylamide groups. The resulting mixture was continued heating overnight(15-20 h) at 65° C. under N₂. The reaction mixture was cooled to roomtemperature and polymer solution was precipitated in 100 ml diethylether, centrifuged, decanted, and the residue was flushed with N₂. Thedeprotection of Boc-amine was carried out by dissolving protectedpolymer in 2 ml of TFA/TIS (triisopropylsilane)/H₂O 95/2.5/2.5 solutionfor 30-60 min at ice bath temperature. The reaction mixture was allowedto warm up to room temperature. The crude polymer was precipitated outagain in 100 ml diethyl ether, centrifuged, decanted, and the residuewas flushed with N₂. The residue was dissolved in 10 ml DI water and thepH was adjusted to pH 7 with 1N NaOH solution. The polymer solution wasthen transferred in to 2K dialysis bag and dialyzed with Milli-Q waterfor 24-48 h and then lyophilized.

wherein the polymer is random or block and wherein n is independently 0to 60; R is C₄H₁₀N; and R′ is hydrogen.

1H and 13C NMR spectra were recorded on varian spectrometer operating at500 MHz. 1H NMR spectra were in full accordance with the expectedstructures. No signals were present in the region between 5 and 7 ppm,corresponding to the acrylamide group, indicating that these polymershave capped end groups. Feed ratios of different monomers in thepolymers were confirmed by NMR. All NMR spectra were taken in deuteratedmethanol.

The molecular weight and polydispersity (Mw/Mn) of the synthesizedpolymers were determined by GPC relative to polystyrene standards(Sigma-Aldrich) using a Waters 2695, Waters 2414 RI detector and TSK-GELAlpha-3000 column. Polymers have Mn between 5 K to 25 K with PDI=1.3-2.8.

Polystyrene Standards (Sigma-Aldrich)

The following polymers were prepared according to the General ReactionScheme and Schemes above.

Polymers

Polymers of the instant invention include:

wherein the polymer is random or block and wherein n is independently 0to 60; R is C₄H₁₀N; and R′ is hydrogen.

Table 1 demonstrates specific random or block polymers. In Table 1,aminoethoxy is always designated as 100, which means that aminoethoxy ispresent in each repeating unit of the polymer.2-(2-aminoethoxy)ethyl-,2-(1H-imidazol-4-yl)ethyl-, anddodecyl-containing repeating units are randomly distributed andincorporated in the identified % ratios, wherein the ratios are +/−5%.

TABLE 1 2-(1H-imidazol-4- aminoethoxy 2-(2-aminoethoxy)ethyl yl)ethyldodecyl 100 30 50 20 100 40 30 30 100 30 20 50 100 0 20 80 100 70 0 30100 20 80 0 100 40 60 0 100 50 0 50 100 50 20 30 100 50 30 20 100 60 400 100 60 20 20 100 60 0 40 100 70 0 30 100 70 10 20 100 70 20 10 100 8020 0 100 80 10 10 100 80 0 20 100 90 10 0 100 90 0 10 100 100 0 0

General Polymer Conjugation (Scheme 3)

The polymers comprising Formula Z or Z′ and the examples shown abovewere synthesized for use in the following conjugation steps toultimately create the polyconjugates of the instant invention. Thepolymers comprising Formula Z or Z′ and the examples disclosed areuseful in the preparation of polyconjugates which are, in turn, usefulfor the delivery of oligonucleotides, specifically the delivery ofsiRNA. Other methods for the synthesis of polyconjugates are describedin WO2008/022309.

Activation of Polymer and Oligonucleotide (Functionalization)

Amines on the polymers were functionalized, with the activated group, toallow attachment of the oligonucleotide to the polymer. Also, theoligonucleotide was modified to react with activiated group on thepolymer.

Polymer-Oligonucleotide Conjugation

Activated groups on the polymer react with modified oligonucleotide toform polymer-oligonucleotide conjugate.

Free RNA duplex as well as free RNA duplex-dimer was determined byaqueous SEC using a GE Heathsciences Superdex 75HR 10/300 column. Themobile phase was composed of 100 mM Tris with 2M NaCl, pH 8.4. Total RNA(both free and bound) was determined by using Inductively Coupled Plasma(ICP) spectroscopy. Since the RNA is the only phosphorus containingspecies in the formulations, determining the total phosphorus contentcan be used to directly determine the total RNA concentration. Once thefree RNA (duplex and duplex-dimer) and total RNA is determined, theamount of RNA conjugated to the polymer can be calculated (i.e.conjugation efficiency).

Masking of Polymer Conjugate

Polymer siRNA conjugate was masked with carboxy dimethylmaleic anhydrideof N-acetylgalactosamine (CDM-NAG) and carboxy dimethylmaleic anhydrideof polyethylene glycol (CDM-PEG).

Total concentrations of CDM-NAG and CDM-PEG were determined usingreverse-phase HPLC with mobile phases of 0.1% TFA in water and 0.1% TFAin 70/30 methanol:acetonitrile. Rapid demasking of the polymer afterinjection onto the column allows quantitation of CDMs with the polymerremoved using a C18 guard column to prevent chromatographicinterference. Free (i.e. unbound) CDM-NAG and CDM-PEG is analyzed byfirst filtering through a 10K centrifuge filter followed by analysisusing the same reverse-phase HPLC method. Masking Efficiency can becalculated by first calculating the bound RNA, CDM-NAG and CDM-PEG. Thepolymer molecular weight in combination with the total amines availablefor conjugation is then used with the bound ligands to calculate maskingefficiency.

Purification of Polymer Conjugate (Optional)

Tangential flow filtration (TFF) process was used to purify maskedpolymer conjugate formulations of un-incorporated components and toexchange buffer to pharmaceutically acceptable formulation vehicle. TheTFF filter material was made of either modified polyethersulfone (PES)or regenerated cellulose. The selection of molecular weight cutoff forthese membranes was done with efficiency of purification and retentionof polymer conjugate in mind. The processing parameters, including butnot limited to feed pressure, retentate pressure, crossflow rate andfiltrate flux were set to allow reproducibility from batch to batch andlinear scaling of the process. Using the difiltration mode of TFF, thereaction impurities were filtered out into the permeate while theretained polymer conjugate underwent a buffer exchange. The purificationwas done at refrigerated conditions. After TFF, the final product wasconcentrated to 0.4-2.0 mg/ml of siRNA and sterile filtered using a 0.2μm PES syringe filter and stored at −20° C. until use.

Specific Polymer Conjugation (Scheme 4)

The polymers comprising Formula Z or Z′ and the specific examples shownabove were synthesized for use in the following conjugation steps toultimately create the polyconjugates of the instant invention. Thepolymers comprising Formula Z or Z′ and the specific examples disclosedare useful in the preparation of polyconjugates which are, in turn,useful for the delivery of oligonucleotides, specifically the deliveryof siRNA. Other methods for the synthesis of polyconjugates aredescribed in WO2008/022309.

Activation of Polymer and Incorporation of siRNA (Functionalization)

About 2.6 mg of polymer in a 4 mL vial is added with ˜87 μL of 100 mMTRIS, 5% glucose pH 9 buffer and stirred until the polymer is dissolved.To this solution was added 3.9 μL of (4-succinimidyloxycarbonyl-

-methyl-

-[2-pyridyldithio]toluene) solution (1 mg/100 μl in DMSO) correspondingto 1.5 wt % with respect to the polymer weight.

5′-C6-amine modified siRNA (1 g, 0.0714 mmol) is dissolved in 0.1Msodium bicarbonate buffer (20 ml, 50 mg/mL) in a vial with magnetic stirbar and cooled to 0-5° C. in an ice water bath. In a separate vialN-Succinimidyl-S-acetylthioacetate (SATA) (83 mg, 0.357 mmol, 5equivalents) is dissolved in 0.78 ml DMSO. The SATA solution is addedover 1 min and the clear, colorless reaction mixture stirred at 0-5° C.for 2 h. After 2 h, the reaction mixture is sampled and analyzed by UPLCor HPLC for completion of the conjugation. If >5% siRNA remainsunreacted, another charge of SATA in DMSO (2.0 equivalents) is added andthe reaction aged at 0-5° C. for completion of the SATA conjugation(confirmation by HPLC or UPLC). When there is <5% unreacted siRNAremaining by UPLC or HPLC, the reaction mixture is purified by TFFdialysis using water (˜2 L) or PD10 column to remove any remainingSATA/succinimides. The recovered purified solution is lyophilized to awhite fluffy solid. The recovery is typically around 95% and the purityis greater than 70% by UPLC.

Polymer-siRNA Conjugation

The activated polymer is diluted with 100 mM TRIS 5% glucose buffer pH 9resulting in a final polymer concentration of ˜2.4 mg/mL. About 0.4 mgof siRNA is added to the acvitated polymer solution and stirred at roomtemperature for one hour and preceded to final masking step. As shown inFIGS. 2, 3 and 4 polyconjugates have polymer siRNA conjugationefficiency >85%.

Masking of Polymer Conjugate

In a vial, 0.4 mg of CDM-PEG is weighed and 0.775 mg of CDM-NAG is addedto this. The siRNA-polymer conjugate solution is then transferred intothis vial containing CDM-PEG and CDM-NAG and stirred for 1 hr at roomtemperature. Measured masking efficiency of polyconjugates is shown inFIGS. 2, 3 and 4.

wherein the polymerconjugate is random or block and wherein n isindependently 0 to 60; R is C₄H₁₀N; and R′ is hydrogen.

As a polymer is random, siRNA can conjugate any pendent primary amineand CDM's can mask any primary amine, for example n is independently 1to 60 and Table 2 demonstrates substituents in the identified % ratios,wherein the ratios are +/−5%.

Polyconjugates

wherein the polymerconjugate is random or block and wherein n isindependently 0 to 60; R is C₄H₁₀N; and R′ is hydrogen.

Table 2 demonstrates specific random or block polymerconjugates. InTable 2, aminoethoxy is always designated as 100, which means thataminoethoxy is present in each repeating unit of the polymerconjugate.2-(2-aminoethoxy)ethyl-, 2-(1H-imidazol-4-yl)ethyl-, anddodecyl-containing repeating units are randomly distributed andincorporated in the identified % ratios, wherein the ratios are +/−5%.

TABLE 2 2-(1H- 2-(2- imidazol- aminoethoxy aminoethoxy)ethyl 4-yl)ethyldodecyl Polyconjugate 100 40 30 30 1 Polyconjugate 100 30 50 20 2Polyconjugate 100 30 20 50 3 Polyconjugate 100 0 20 80 4 Polyconjugate100 70 0 30 5 Polyconjugate 100 20 80 0 6 Polyconjugate 100 40 60 0 7

EXAMPLE 1

RBC Hemolysis Assay:

Human blood was collected in 10 ml EDTA Vacutainer tubes. A smallaliquot was assessed for evidence of hemolysis by centrifugation at15000 RCF for 2 min and non-hemolyzed samples were carried forward intothe assay. Red blood cells (RBCs) were washed three times in either 150mM NaCl/20 mM MES, pH 5.4, or 150 mM NaCl/20 mM HEPES, pH 7.5 bycentrifuging at 1700×g for 3 min and resuspending in the same buffer toyield the initial volume. RBCs were then diluted in appropriate pHbuffer to yield 10⁸ cells in suspension. A 10× stock concentration ofeach test agent (Polymerconjugate 1, Polymerconjugate 2) was preparedand a 10 point, 2-fold dilution was performed in appropriate pH buffers.The diluted test agents were added to the RBCs in appropriate pH buffersin Costar 3368 flat-bottom 96 well plates. Solutions were mixed 6 to 8times and the microtiter plate was covered with a low evaporation lidand incubated in a 37° C. warm room or incubator for 30 minutes toinduce hemolysis. The plate was then centrifuged at 1700×g for 5 min and150 μl supernatants were transferred to a Costar 3632 clear bottom 96well plate. Hemoglobin absorbance was read at 541 nM using a TecanSafire plate reader and percent hemolysis was calculated assuming 100%lysis to be measured by the hemoglobin released by RBCs in 1% TritonX-100.

As shown in FIG. 1, polymerconjugates masked with acid labile maskingagents at pH 7.4 don't show any lytic activity, however, become lytic atpH 5.4.

EXAMPLE 2

HepG2 Gene Silencing and Toxicity Data:

HepG2 cells were plated in 96-well microtiter plates at 6000 cells/welland incubated overnight at 37° C. to allow cell adherence. 10× stock ofPCs (polyconjugates) were prepared in media and 20 μl 10× PC was addedto 180 μl media already in wells resulting in 1× final treatment and a300-0 nM 10-point half log titration, based on siRNA concentration.Cells were incubated with PCs in 37 degrees CO₂ incubator for 24-72 h.MTS Toxicity Assay was performed on 24 h-72 h treated cells andcytotoxicity was assessed by CellTiter 96 Aqueous One Solution CellProliferation Assay (Promega #G3581, Madison, Wis.). 40 μl MTS Solutionwas added, incubated in 37 degrees CO₂ incubator 1 hour, absorbance at490 nm was read on Tecan Safire. Cells were then washed 3× in PBS and150 μl/well bDNA DLM Lysis Buffer (Panomics “Quantigene” 1.0 bDNA kit#QG0002, Fremont, Calif.) was added. Plate was then incubated at 37degrees in Warm Room 30 min. Lysates were removed and frozen at −70degrees C. overnight. The next day, all cell lysates were thawed at RTand 20 μl of each lysate was removed and used for determination of totalprotein using Micro BCA Protein Assay kit (Pierce #23235, through ThermoScientific, Rockford, Ill.). Absorbance was measured on Tecan Safire:Wavelength=562 nM, Plate=Costar96 ft, Number of Reads=100, Time betweenReads=5.50 μl each lysate was also used to determine mRNA expressionlevels in cells treated with SSB siRNA.

ApoB mRNA knockdown was determined using Quantigene 1.0 bDNA Assay(Panomics # QG0002 Lot # 51CW36, Fremont, Calif.), a kit designed toquantitate RNA using a set of target-specific oligonucleotide probes.

Active siRNA 1: Zimmerman Apo B:

Zimmermann et al., (2006) Nature, 441(7089):111-114 ordoi:10.038/nature04688 (see supplementary information).

Active siRNA 2: Sci 10 Apo B:

Sci10 ApoB siRNA (SEQ ID NO.: 1) 5′-iB-CUUU AA C AA UUCCU GAAA UTsT-iB-3′ (SEQ ID NO.: 2) 3′-UsU GAAA U UG UU AAGGA CUsUsUsA-5′ U -Ribose iB - Inverted deoxy abasic AGU - 2′ Fluoro T - 2′ Deoxy CU - 2′OCH₃ s - phophorothioate linkage

The passenger strand contains a primary amine with six carbon linker at5′ end, which is used to conjugate the siRNA to the polymer.

Control siRNA: Low Hex 9:

Low Hex 9 siRNA (SEQ ID NO.: 3) 5′-amil-iB-CU AG CU GGA C A C G UC GA UATsT-iB-3′ (SEQ ID NO.: 4) 3′-UsUGA UC GACCU G U G C AG CUAU-5′ amil -amino linker iB - Inverted deoxy abasic CU - 2′-Fluoro (F) AGT -2′-Deoxy UGA - 2′-Methoxy (OMe) AU - Ribose s - phosphorothioate linkage

The passenger strand contains a primary amine with six carbon linker at5′ end, which is used to conjugate the siRNA to the polymer.

Panomics Quantigene bDNA Kit # QG0002-Protocol for 96 Well Plate:

Day 1

Make diluted lysis mixture (DLM) by mixing 1 volume of lysis mixturewith 2 volumes of Nuclease Free water (Ambion cat # AM9930). Aspirate(PBS) from plate. Add 150 μl DLM to each well and mix. (Include Column 1as Buffer Alone Background). Incubate at 37° C. for 30 minutes. (Afterheating, Lysates can be placed in the −70° C. freezer until analysis isperformed. If lysates are frozen, thaw at Room Temperature and incubateat 37° C. for 30 minutes and mix well before adding the samples to thecapture plate.) Bring all reagents to Room Temperature before use,including the capture plates. Dilute CE, LE and BL probe set components:0.1 μl well each into DLM. Add (100-X) μl diluted probe set/well. Add(X) μl cell lysate/well. Cover with foil plate sealer. Incubate at 53°C. for 16-20 hrs. Note: If assay contains multiple plates, perform steps7, 8, 9 on 2-3 plates at a time and place at 53° C. before going on tonext 2-3 plates.

Day 2

Bring Amplifier, Label Probe and Substrate to Room Temperature. Vortexand briefly centrifuge the tubes of Amplifier and Label Probe to bringthe contents to the bottom of the tube. Prepare Wash buffer: add 3 mlComponent 1 and 5 ml Component 2 to 1 L distilled water. (Wash Buffer isstable at Room Temperature for up to 6 months)

Prepare as needed: Amplifier Working solution, Label Probe WorkingSolution, and Substrate

Working Solution:

-   Amplifier Working Solution—1:1000 dilution into Amplifier/Label    Probe diluent.-   Label Probe Working solution—1:1000 dilution into Amplifier/Label    Probe diluent.-   Substrate Working Solution—1:333 dilution of 10% Lithium Lauryl    Sulfate Substrate into Substrate Solution (protect from light).

Add 200 μl/well of wash buffer to overnight hybridization mixture.Repeat washes 3× with 300 μl of Wash Buffer. *Do not let the captureplates stand dry for longer than 5 minutes. Add 100 μl/well of AmplifierWorking Solution. Seal plate with clear seal and incubate at 53° C. for30 minutes. Wash plate 3× with 300 μl of Wash Buffer. Add 100 μl well ofLabel Probe Working Solution. Seal plate with clear seal and incubate at53° C. for 30 minutes. Wash plate 3× with 300 μl of Wash Buffer. Add 100μl/well Substrate Working Solution. Seal plate with foil seal andincubate at 53° C. for 15 minutes. Let plate stand at Room Temperaturefor 10 minutes. Read in luminometer with integration time set to 0.2seconds. bDNA data was normalized to protein and graphed using GraphPadPrism Program using non-linear regression curve fit analysis.

As shown in Table 3, all the polyconjugates are active in vitro.

TABLE 3 bDNA (nM) MTS (nM) Polyconjugate 1 126 >300 Polyconjugate 2112 >300 Polyconjugate 3 135 >300 Polyconjugate 4 11 13 Polyconjugate 5148 >300 Polyconjugate 6 53 >300 Polyconjugate 7 59 >300In Vivo Evaluation of Efficacy (Mice):

CD1 mice were tail vein injected with the siRNA containing polymerconjugates at a dose of 3 mg/kg in a volume of 0.2 mL, 100 mM TRIS/9%glucose, pH9, vehicle. Five days post dose, mice were sacrificed andliver tissue samples were immediately preserved in RNALater (Ambion).Preserved liver tissue was homogenized and total RNA isolated using aQiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit followingthe manufacturer's instructions. Liver ApoB mRNA levels were determinedby quantitative RT-PCR. Message was amplified from purified RNAutilizing primers against the mouse ApoB mRNA (Applied Biosystems Cat.No. Mm01545156_m1). The PCR reaction was run on an ABI 7500 instrumentwith a 96-well Fast Block. The ApoB mRNA level is normalized to thehousekeeping PPIB mRNA and GAPDH. PPIB and GAPDH mRNA levels weredetermined by RT-PCR using a commercial probe set (Applied BiosytemsCat. No. Mm00478295_m1 and Mm4352339E_m1). Results are expressed as aratio of ApoB mRNA/PPIB/GAPDH mRNA. All mRNA data is expressed relativeto the vehicle control.

As shown in FIG. 2, mice treated with active Apo B siRNA polyconjugatesshow significant reduction in mRNA expression, whereas no reduction inmRNA expression was observed with control siRNA polyconjugates.

Polymer driven knockdown (KD) was observed with polymerconjugate 1 asshown in FIG. 2: ID-B and ID-E. The amount of polymer used in both casesis the same while the siRNA doses are different (24 & 3 mpk for E and 24& 6 mpk for B). Both of these conjugates gave same KD.

Dose depended KD was observed with polyconjugate 1 and 2 at 6, 3 and 1mpk dose.

As shown in FIG. 3, all the polyconjugates (3, 4, 5, 6 and 7) are activein vivo and the percentage of KD varies with the change in incorporationratios of monomers.

In Vivo Evaluation of Efficacy (Rats)

Polyconjugates were dosed by tail vein injection into female SpragueDawley rats (150-200 grams) at a rate of 3 ml/min. Five days post dose,rats were sacrificed and liver tissue samples were immediately preservedin RNALater (Ambion). Preserved liver tissue was homogenized and totalRNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNAisolation kit following the manufacturer's instructions. Liver ApoB mRNAlevels were determined by quantitative RT-PCR. Message was amplifiedfrom purified RNA utilizing primers against the mouse ApoB mRNA (AppliedBiosystems Cat. No. Mm01545156_m1). The PCR reaction was run on an ABI7500 instrument with a 96-well Fast Block. The ApoB mRNA level isnormalized to the housekeeping PPIB mRNA and GAPDH. PPIB and GAPDH mRNAlevels were determined by RT-PCR using a commercial probe set (AppliedBiosytems Cat. No. Mm00478295_m1 and Mm4352339E_m1). Results areexpressed as a ratio of ApoB mRNA/PPIB/GAPDH mRNA. All mRNA data isexpressed relative to the vehicle control. Alanine aminotransferanse(ALT) was measured using the ADVIA Chemistry Systems AlanineAminotransferase (ALT) method, 03815151, Rev. A., according to thefollowing reference, Clinical and Laboratory Standards Institute.Laboratory Documents: Development and Control; Approved Guideline—FifthEdition. CLSI document GP2-A5 [ISBN 1-56238-600-X]. Clinical andLaboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,Pa., 19807-1898 USA, 2006.

As shown in FIG. 4, dose depended KD was observed with polyconjugate 2at 6, 3 and 1 mpk dose in rats with 2 times elevation in ALT.

What is claimed is:
 1. A polymer comprising Formula Z:

wherein: n is 2 to 250; R¹ is independently selected from the groupconsisting of a primary amine, secondary amine, tertiary amine, andquaternary amine; and R² is independently selected from the groupconsisting of a primary amine, secondary amine, tertiary amine,quaternary amine, a heterocyclic amine, and a lipophilic group; orstereoisomer thereof.
 2. A polymer according to claim 1 comprisingFormula Z′:

wherein: n is 2 to 250; R is a capped end group selected from the groupconsisting of a primary amine and secondary amine; R′ is hydrogen ormethylene; R¹ is independently selected from the group consisting of aprimary amine, secondary amine, tertiary amine, and quaternary amine;and R² is independently selected from the group consisting of a primaryamine, secondary amine, tertiary amine, quaternary amine, a heterocyclicamine, and a lipophilic group; or stereoisomer thereof.
 3. A polymeraccording to claim 1 comprising Formula Z′, wherein: n is 2 to 250; R isC₄H₁₀N; R′ is hydrogen; R¹ is aminoethoxy; and R² is independentlyselected from dodecyl, 2-(1H-imidazol-4-yl)ethyl and2-(2-aminoethoxy)ethyl; or stereoisomer thereof.
 4. A polymer accordingto claim 1 which is selected from:

wherein the polymer is random or block and wherein n′, n″, and n′″ areindependently 0 to 60, provided that the sum of n′, n″, and n′″ is atleast 2; R is C₄H₁₀N; and R′ is hydrogen.
 5. A polymer conjugatecomposition comprising a polymer according to claim 1 of Formula Z, alinker and an oligonucleotide.
 6. A polymer conjugate composition ofclaim 5 further comprising a masking agent.
 7. A polymer conjugatecomposion of claim 5 further comprising a targeting ligand.
 8. A polymerconjugate composition of claim 5 further comprising a masking agent anda targeting ligand.
 9. A polymer conjugate composition made by the 1)synthesis of an activated polymer comprising Formula Z:

wherein: n is 2 to 250; R¹ is independently selected from the groupconsisting of a primary amine, secondary amine, tertiary amine, andquaternary amine; and R² is independently selected from a primary amine,secondary amine, tertiary amine, quaternary amine, a heterocyclic amine,and a lipophilic group; or stereoisomer thereof; 2) synthesis of anactivated oligonucleotide; and 3) conjugation of the activated polymerwith the activated oligonucleotide; optionally including the addition ofa masking agent and/or a targeting ligand.